Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the co-clustering with these proteins on the neuronal cell membranes remain elusive at the electron microscopic level. In this study, we quantitatively investigated the nanoscale distribution of PI(4,5)P2 on the neuronal cell membranes by SDS-digested freeze-fracture replica labeling with cryo-fixed mouse cerebellum. We demonstrate that PI(4,5)P2 makes clusters with a mean size of [~] 1,000 nm2 and these clusters show preferential accumulation in specific membrane compartments depending on cell types. Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones are particularly rich in the PI(4,5)P2 clusters. Furthermore, these clusters are extensively associated with clusters of CaV2.1 and GIRK3 throughout different membrane compartments of PCs, GCs, and molecular layer interneurons. In contrast, these clusters showed compartment-specific association with mGluR1 in PC spines. These results suggest that visualization of the nanoscale PI(4,5)P2 distribution may provide insight into the physiological functions of PI(4,5)P2 in neurons.